DESCRIPTION (Adapted from the applicant's): The 234 residue leucyl/phenylalanyl-tRNA-protein transferase (L/F transferase) catalyzes the transfer of leucyl or phenylalanyl moieties from their aminoacyl- tRNAs to the amino terminus of acceptor proteins. This peptidyl- transferase reaction, which is energetically identical to the reaction catalyzed by the ribosome, serves as a powerful degradation signal via the N-end rule pathway in E. coli cells. Specific Aim 1: To explore the mechanism of aminoacyl-tRNA recognition and acceptor protein recognition by the L/F transferase. Recognition of aminoacyl-tRNAs is a process central to protein synthesis. The L/F transferase provides a simplified system for the study of aminoacyl-tRNA and peptide recognition. Investigators will use a combination of genetic and biochemical methods in order to determine how the L/F transferase is able to recognize a specific subset of aminoacyl-tRNAs. The ability to utilize multiple types of aminoacyl-tRNAs is shared by the cell's translation machinery making these results of fundamental importance. The regions of the L/F-transferase responsible for acceptor protein recognition will be identified genetically through the isolation and sequence analysis of mutant enzymes with relaxed specificities. Enzymes with relaxed acceptor protein specificities are expected to be useful as reagents in studies requiring end-labeled proteins and as diagnostic reagents. Experiments are proposed that are designed to detect increased structure of the total enzyme induced by aminoacyl-tRNA binding. These results will be used to begin to separate the regions of the enzyme responsible for substrate recognition and catalysis. Specific Aim 2: To understand the physiological role of the N-end rule pathway of protein degradation to determine the cellular fate and identity of the natural substrates of the L/F-transferase. The determinants of proteins which result in cellular instability are often complex and are generally poorly understood. The L/F-transferase adds a single amino acid to the N-terminus of an engineered acceptor protein which results in a ~1000-fold increase in the degradation of the substrate. Identification of natural L/F-transferase substrates has been very elusive as has been the identification of the cellular processing enzymes responsible for the generation of unusual N-terminal residues in cells. This application describes systematic approaches to identify the natural substrates of the L/F-transferase in order to determine the cellular function of the complex N-end rule pathway.